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棉花学报 ›› 2016, Vol. 28 ›› Issue (2): 129-137.doi: 10.11963/issn.1002-7807.201602005

• 研究与进展 • 上一篇    下一篇

棕色棉茎尖基因枪遗传转化

侯云鹏,冯鸿杰,程文瀚,张新宇,孙杰,刘永昌*   

  1. 石河子大学农学院/新疆兵团绿洲生态农业重点实验室,新疆 石河子 832003
  • 收稿日期:2015-09-28 出版日期:2016-03-15 发布日期:2016-03-15
  • 通讯作者: liuyongchang2003@126.com
  • 作者简介:侯云鹏(1990―),男,硕士研究生,1351904022@qq.com
  • 基金资助:
    国家自然科学基金——原花青素在棕色棉纤维颜色形成中的作用及分子机制研究(U1303281);国家科技重大专项——转基因生物新品种培育(2011ZX08005-005);石河子大学动植物育种专项(GXJS2012-YZ01)

Biolistic Genetic Transformation of Brown Cotton Shoot Tips

Hou Yunpeng, Feng Hongjie, Cheng Wenhan, Zhang Xinyu, Sun Jie, Liu Yongchang*   

  1. College of Agricultural, Shihezi University / Oasis Eco-Agricultural Key Laboratory, The Xinjiang Production and Construction Crops, Shihezi, Xinjiang 832000, China
  • Received:2015-09-28 Online:2016-03-15 Published:2016-03-15

摘要: 为了建立高效的棉花茎尖遗传转化体系,以棕色棉品种新彩棉5号为材料,采用茎尖培养和基因枪遗传转化方法,通过对凝固剂种类、活性炭含量、卡那霉素浓度、渗透处理时间、轰击压力、轰击距离等因素的优化,建立了棕色棉新彩棉5号茎尖基因枪遗传转化技术体系。结果表明,不同凝固剂种类、活性炭含量、卡那霉素浓度、渗透处理时间、轰击压力和轰击距离对棕色棉茎尖转化率有明显影响。最佳的遗传转化体系为:前渗处理4 h,轰击压力7.586 MPa(1100 psi),轰击距离9 cm,轰击后恢复培养4 d,然后在MSB+8 g·L-1琼脂+125 mg·L-1卡那霉素筛选培养基中筛选25 d。获得抗性芽后,在添加1 g·L-1活性炭的生根培养基中诱导抗性芽生根,并获得抗性再生植株。通过PCR和qPCR检测,5200个茎尖共获得16株阳性转基因植株,转化率为0.31%。该技术体系的建立为棕色棉的遗传转化奠定了基础,为彩色棉育种提供了新的材料。

关键词: 棕色棉; 茎尖; 基因枪遗传转化; 转化率

Abstract: To genetically transform cotton shoot tips, we established a biolistic genetic transformation system though the plant shoot tip culture technique and the gene gun-mediated transformation method, using the shoot tips of brown cotton Xincaimian No.5, a Gossypium hirsutum L. variety cultured in Xinjiang as materials. Several critical factors of the system were assessed, including the type of coagulant, the activated charcoal concentration, the kanamycin concentration, the penetration processing time, and the bombardment pressure and distance. Results showed that each factor clearly influenced the transformation rate of brown cotton shoot tips. An optimal biolistic genetic transformation technology system was determined: (1) shoot tips were cultured for 4 h before being bombarded in the osmotic medium; (2) the shoot tips were bombarded with helium pressure of 7.586 MPa (1100 psi), and a bombardment distance of 9 cm; (3) after culturing for 4 d in the recovery medium, shoot tips were moved to MSB screening medium containing 8 g·L-1 agar powder and 125 mg·L-1 kanamycin and then screened for 25 d to obtain the resistant germs; (4) then, 1 g·L-1 activated charcoal was added to the induction medium to induce rooting of the resistant germs to obtain resistant regenerating plants. After PCR and qPCR detections, 16 plants positive for the transgene were obtained from 5200 shoot tips, with a transformation rate of 0.31%. The establishment of this technology system lays a foundation for the genetic transformation of brown cotton and provides new materials for naturally-colored cotton breeding.

Key words: brown cotton; shoot tips; gene gun-mediated transformation method; transformation rate

中图分类号: 
  • S562.03